Enzyme replacement therapy (ERT) has been successfully implemented to treat subjects with deficiencies in alkaline phosphatase (AP) activity. In particular, such therapies are useful for treating bone mineralization defects associated with deficient AP activity. Several factors regulate bone formation and resorption, including, for example, serum calcium and phosphate concentrations, and circulating parathyroid hormone (PTH). FGF23, for example, is a hormone that contributes to the regulation of calcium and phosphate homeostasis-promoting renal phosphate excretion and reducing circulating levels of active vitamin D (diminishing intestinal absorption of calcium). ERT treatment that leads to normalized bone formation can potentially have an effect on the production of modulators (e.g., hormones such as, for example, parathyroid hormone (PTH), or vitamin D) that regulate or are regulated by bone mineralization factors (e.g., serum calcium and phosphate).
PTH, also referred to as “parathormone” or “parathyrin,” is secreted by the parathyroid gland as an 84-amino acid polypeptide (9.4 kDa). PTH acts to increase the concentration of calcium (Ca2+) in the blood by acting upon the parathyroid hormone 1 receptor (high levels of the parathyroid hormone 1 receptor are present in bone and kidney) and the parathyroid hormone 2 receptor (high levels of the parathyroid hormone 2 receptor are present in the central nervous system, pancreas, testes, and placenta).
PTH enhances the release of calcium from the large reservoir contained in the bones by affecting bone resorption by modulation of expression of key genes that regulate bone resorption and formation. Bone resorption is the normal degradation of bone by osteoclasts, which are indirectly stimulated by PTH. Since osteoclasts do not have a receptor for PTH, PTH's effect is indirect, through stimulation of osteoblasts, the cells responsible for creating bone. PTH increases osteoblast expression of the receptor activator of nuclear factor kappa-B ligand (RANKL) and inhibits the expression of osteoprotegerin (OPG). OPG binds to RANKL and blocks it from interacting with RANK, a receptor for RANKL. The binding of RANKL to RANK (facilitated by the decreased amount of OPG available for binding the excess RANKL) stimulates fusion of osteoclasts into multinucleated osteoclasts, ultimately leading to bone resorption. The downregulation of OPG expression thus promotes bone resorption by osteoclasts.
PTH production (synthesis of PTH) is stimulated with high serum levels of phosphates (often present in late stages of chronic kidney disease) by direct effect of serum phosphates on PTH synthesis in the parathyroid gland by promoting the stability of PTH. PTH negatively impacts retention of phosphates in kidneys (promoting loss through urine) affecting homeostasis of phosphates and calcium. The importance of this signaling pathway in the renal response to PTH is highlighted by the renal resistance to PTH associated with deficiency of PTH receptor G protein subunit (Gsalpha) deficiency in patients with pseudohypoparathyroidism. PTH also enhances the uptake of phosphate from the intestine and bones into the blood. In the bone, slightly more calcium than phosphate is released from the breakdown of bone. In the intestines, absorption of both calcium and phosphate is mediated by an increase in activated vitamin D. The absorption of phosphate is not as dependent on vitamin D as is that of calcium. The end result of PTH release from the parathyroid gland is a small net drop in the serum concentration of phosphate.
Secretion of PTH is controlled chiefly by serum Ca2+ through negative feedback. Increased levels of calcium reduce PTH secretion, while diminished levels increase PTH secretion. Calcium-sensing receptors located on parathyroid cells are activated when Ca2+ is elevated. G-protein coupled calcium receptors bind extracellular calcium and are found on the surface of a wide variety of cells distributed in the brain, heart, skin, stomach, parafollicular cells (“C cells”), and other tissues. In the parathyroid gland, high concentrations of extracellular calcium result in activation of the Gq G-protein coupled cascade through the action of phospholipase C. This hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to liberate intracellular messengers IP3 and diacylglycerol (DAG). Ultimately, these two messengers result in a release of calcium from intracellular stores and a subsequent flux of extracellular calcium into the cytoplasmic space. The effect of this signaling of high extracellular calcium results in an intracellular calcium concentration that inhibits the secretion of preformed PTH from storage granules in the parathyroid gland. In contrast to the mechanism that most secretory cells use, calcium inhibits vesicle fusion and release of PTH.
Additional mechanisms that affect the amount of PTH available for secretion involve, for example, calcium-sensitive proteases in the storage granules. Upon activation increase the cleavage of PTH (1-84) into carboxyl-terminal fragment, further reducing the amount of intact PTH in storage granules.
PTH also increases the activity of 1-α-hydroxylase enzyme, which converts 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol, the active form of vitamin D in kidneys. Vitamin D decreases transcription of the PTH gene. Vitamin D deficiency (often seen in chronic renal disorders) thus causes increases in PTH production. FGF23 is another regulator of parathyroid function, it is secreted by osteocytes or osteoblasts in response to increased oral phosphate intake and other factors. It acts on kidney to reduce expression transporters of phosphates in kidney reducing phosphate retention. In early stages of chronic renal disease, levels of FGF23 are increased to help promote the urinary excretion of phosphates. Elevated FGF23 in chronic renal disorders reduces activity of the Vitamin D 1-α-hydroxylase enzyme and results low production of the active form of vitamin D. In the intestines, absorption of calcium is mediated by an increase in activated vitamin D. Diminished intestinal calcium absorption, which leads to serum hypocalcemia, does not provide strong negative feedback to production/release of PTH from parathyroid gland, causing increased release of PTH from the parathyroid gland. FGF23 appears to directly inhibit PTH secretion as well.
As AP replacement therapy replaces part of a complex pathway, for example, for proper bone formation, there is a need to further characterize the pathway, and to identify analytes that are indicative of therapeutic effects. Such tracking may indicate therapeutic efficacy and/or may identify additional therapies that may become necessary as a result of AP replacement therapy.